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*
Department of Immunology and Bacteriology, University of Glasgow, and
Department of Immunology, University of Strathclyde, Glasgow, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/LPS-induced production, by
macrophages, of bioactive IL-12 (p70), a key cytokine in the
development of Th1 responses. This suppression of the induction of a
component of the host immune response extends to the production of the
proinflammatory cytokines IL-6 and TNF-
, but not NO. The molecular
mechanism underlying these findings awaits elucidation but,
intriguingly, the initial response of macrophages to ES-62 is to
demonstrate a low and transient release of these cytokines before
becoming refractory to further release induced by IFN-/LPS. The
relevance of our observations is underscored by the finding that
macrophages recovered from mice exposed to "physiological" levels
of ES-62 by the novel approach of continuous release from implanted
osmotic pumps in vivo were similarly refractory to release of IL-12,
TNF-, IL-6, but not NO, ex vivo. Therefore, our results suggest that
exposure to ES-62 renders macrophages subsequently unable to produce
Th1/proinflammatory cytokines. This likely contributes to the
generation of immune responses with an anti-inflammatory Th2
phenotype, a well-documented feature of filarial nematode
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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140
million people are infected with these three parasites, and another
1000 million are at risk (1). Infection with filarial nematodes is normally lifelong, and individual adult worms can live for >5 years (2). This longevity may, at least in part, reflect the ability of the parasite to induce defects in the host immune system, incorporating modulation of parasite-specific and even more generalized B and T cell responses (reviewed in Refs. 3, 4, 5). Filarial nematodes release a number of proteins into their environment, some of which are biologically active and considered to play roles in the maintenance of infection and parasite survival (6). Excretory-secretory (ES)3 products released by worms are found in the bloodstream of infected humans and animals where they have ample opportunity to interact with host lymphocytes (7, 8). Consistent with this, sera from infected humans or animals containing ES molecules released by the worms exhibit immunosuppressive properties (9, 10). Furthermore, ES products have been demonstrated to inhibit lymphocyte proliferation in vitro (11, 12).
ES-62, a major ES product of the rodent filarial nematode Acanthocheilonema viteae, is a 62-kDa, phosphorylcholine (PC)-containing glycoprotein (reviewed in Refs. 13, 14). We have previously demonstrated the ability of ES-62 to inhibit B and T lymphocyte activation via their Ag receptors (11, 12, 15, 16). However, ES-62 also possesses the capacity to polarize the nature of an immune response. For example, ES-62 generates a Th2 Ab response in vivo resulting in increased serum levels of ES-62-specific IgG1 but not IgG2a, and this skewing of the immune response appears to be mediated by IL-10 (17).
Macrophages play key roles in directing the host immune response to
infection. Recruitment and stimulation of macrophages by cytokines
and/or microbial products such as LPS results in the release of several
key immune effector molecules, such as IL-12, IL-6, TNF-, and NO.
These immune mediators play crucial roles in the development of
immunity against a variety of pathogens. We now report that although
treatment of murine macrophages with ES-62 briefly stimulates
production of low levels of IL-12, IL-6, and TNF-, such cells
subsequently show a substantially reduced capacity to produce these
cytokines when exposed to their classic inducers IFN- and LPS. We
also demonstrate that this suppression can be achieved by in vivo
exposure to ES-62 released from osmotic pumps. These results suggest
that ES-62-mediated suppression of the production of proinflammatory
cytokines from macrophages may contribute to the immunomodulatory
properties of ES-62 that drive the generation of immune responses with
an anti-inflammatory and/or Th2 phenotype.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents used were obtained from Sigma (Poole, U.K.) unless
indicated otherwise; BALB/c and 129 mice (8-wk old) were obtained from
Harlan Olac (Bicester, U.K.). Purified ES-62 from A. viteae
was prepared as described previously (11). Abs against
murine cytokines were obtained from the following sources:
anti-IL-12 p40 and anti-IL-6 Ab pairs, and anti-IL-12 p70
and anti-TNF- ELISA kits from PharMingen (San Diego, CA).
HRP-conjugated anti-rabbit IgG Ab was obtained from Diagnostics
Scotland (Carluke, U.K.).
Purification of murine peritoneal macrophages, cell culture, and cytokine, and NO2- measurement
Thioglycollate-elicited peritoneal macrophages were removed from
BALB/c or 129 mice by peritoneal washing and enriched by plastic
adherence. Adherent peritoneal macrophages were cultured at 37°C/5%
CO2 in DMEM (complete DMEM; Life Technologies,
Paisley, U.K.) supplemented with 10% heat-inactivated FCS (Life
Technologies), 2 mM L-glutamine, 50 U/ml penicillin, and 50
µg/ml streptomycin. Cells were cultured in 96-well plates and
incubated with or without 2 µg/ml ES-62 for 18 h before
stimulation with 100 U/ml IFN- + 100 ng/ml
Salmonella minnesota LPS (Sigma). Culture
supernatants were collected 24 h later and assayed for NO
production by Griess reaction or cytokines by ELISA as previously
described (18). Identical results were obtained with
resident and thioglycollate-elicited macrophages (results not
shown).
MTT assay
Following removal of culture supernatants for cytokine analysis, cell viability was assessed by replacing medium and adding 500 µg/ml MTT reagent (Sigma). After 3 h at 37°C all medium was removed, the precipitate dissolved in isopropanol, and the OD600 was determined.
TaqMan real-time PCR
TaqMan real-time RT-PCR was performed according to the
manufacturer’s instructions (Applied Biosystems, Foster City,
CA). RNA (1–5 µg) was reverse transcribed using 100 U SuperScript II
RT (Life Technologies) at 42°C for 50 min in the presence of 50 mM
Tris-HCl buffer (pH 8.3) containing 75 mM KCl, 3 mM
MgCl2, 5 mM DTT, 0.5 mM dNTP, and 5 µM
oligo(dT)16 (Life Technologies). Primers and
fluorogenic probes were designed using the PrimerExpress v1.0 program
and purchased from Applied Biosystems. The probes used were 5'
6-carboxy-fluorescein (FAM; reporter) and 3' 6-carboxy-tetramethyl
rhodamine (TAMRA; quencher). Murine (m)IL-12 p40: probe 5'
FAM-AACAAGACTTTCCTGAAGTGTGAAGCACCAAAT-TAMRA 3'; forward primer
5'-GGAATTTGGTCCACTGAAATTTTAAA-3'; reverse primer
5'-CACGTGAACCGTCCGGAGTA-3'. mIL-12 p35: probe 5'
FAM-CAGCACATTGAAGACCTGTTTACCACTGGA-TAMRA 3'; forward primer
5'-AAGACATCACACGGGACCAAA-3'; reverse primer
5'-CAGGCAACTCTCGTTCTTGTGTA-3'. The probes and primers for
mTNF- were provided by Applied Biosystems. PCRs were performed in
the ABI-prism 7700 Sequence Detector, which contains a Gene-AMP PCR
system 9600 (Applied Biosystems). PCR amplifications were performed in
a total volume of 25 µl of 10 mM Tris-HCl buffer (pH 8.3) containing
0.5 µl cDNA sample, 50 mM KCl, 10 mM EDTA, 200 µM dATP, dCTP, dGTP,
and 400 µM dUTP, 5 mM MgCl2, 300 nM each
primer, 0.625 U AmpliTaqGold, and 0.25 U AmpErase Uracil
N-Glycolase (Applied Biosystems). Each reaction also
contained 200 nM detection probe. Each PCR amplification was performed
in triplicate using the following conditions: 2 min at 50°C and 10
min at 94°C followed by 40 or 45 two-temperature cycles (15 s at
94°C and 1 min at 60°C). Data analysis was performed using the
Applied Biosystems Sequence Detection Software, and samples were
normalized by their reference reporter hypoxanthine-guanine
phosphoribosyltransferase (HPRT).
ES-62 pumps
ALZET osmotic pumps (Charles River U.K. Ltd, Margate, U.K.) were used as directed by the manufacturer (see also Ref. 19). Pumps containing ES-62 (in PBS, pH. 7.2) were inserted under the skin on the backs of five 20-wk-old, male BALB/c mice. ES-62 was released at a rate of 0.05 µg/h (unless otherwise indicated) over a 2-wk period. An adult female worm releases ES-62 in vitro at a rate of 0.038–0.092 µg/h (W. Harnett, unpublished results); hence, this is within the range predicted to be released by a single transplanted worm (assuming similar release rates in vivo and in vitro). Five control mice were given PBS-releasing pumps. Mice were then killed, and peritoneal macrophages were removed by peritoneal washing and cultured as above.
Statistics
Statistical significance was analyzed by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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from
macrophages activated by IFN- + LPS
IL-12 and TNF- are produced when murine peritoneal macrophages
are stimulated with IFN- and LPS. To investigate whether ES-62
alters production of these cytokines, thioglycollate-elicited
peritoneal macrophages from BALB/c mice were pretreated with various
concentrations of ES-62 for 18 h before stimulation with IFN-
(100 U/ml) and LPS (100 ng/ml) for 24 h. IL-12 p40 (monomer,
homodimer, bioactive p70 heterodimer) and TNF- levels in 24-h
culture supernatants were then assayed by ELISA. Production of IL-12
p40 and TNF- was inhibited by ES-62 pretreatment in a dose-dependent
manner (Fig. 1
). ES-62 at concentrations of up to 100 ng/ml had little effect on
either IL-12 p40 or TNF- produced in response to LPS/IFN-, but
pretreatment with 1–5 µg/ml ES-62 effectively abolished such IL-12
p40 production (Fig. 1A) and reduced TNF- induction by
>50% (Fig. 1B). Therefore, a concentration of 2 µg/ml
ES-62, which has previously been shown to give optimal inhibition of B
cell receptor-mediated cell proliferation (11) and is
within the range of concentrations of PC-containing ES products found
in the bloodstream of filariasis patients (20), was
selected for subsequent experiments.
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+ LPS still resulted in inhibition of
IL-12 p40 production (Fig. 2). Furthermore, transfer of culture supernatant from ES-62-pretreated
cells to resting macrophages just before stimulation with IFN- + LPS
resulted in normal IL-12 p40 production (Fig. 2), suggesting that
suppression occurs as a direct result of ES-62 treatment rather than
via the induction of another inhibitory factor. This proposal is
supported by additional studies showing that shorter periods of
preincubation with ES-62 and even simultaneous addition with IFN- +
LPS also resulted in suppression of macrophage cytokine production
(Fig. 2 (a) and results not shown).
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To further investigate the effects of ES-62 on cytokine production
by macrophages, BALB/c peritoneal macrophages were pretreated with 2
µg/ml ES-62 for 18 h before stimulation with IFN- and/or LPS
or cultured with medium alone for 24 h. IL-12, TNF-, IL-6, and
IL-10 levels were then measured in culture supernatants. Consistent
with previous reports, stimulation of murine peritoneal macrophages
with LPS induced production of IL-12, IL-6, and TNF- (Fig. 3). Costimulation with IFN-, which itself did not stimulate production
of these cytokines, synergistically enhanced IL-12 and IL-6 release
while having no further effect on TNF-. In the absence of LPS
stimulation, i.e., resting or IFN--treated cells, ES-62 induced low
levels of IL-12 p40, IL-6, and TNF- (Fig. 3, A, C, and
D). In contrast, ES-62 pretreatment resulted in 94, 72, and
57% inhibition of IFN- + LPS-induced IL-12 p40, IL-6, and TNF-
production, respectively; LPS-induced TNF- was also reduced by 63%
(Fig. 3, A, C, and D). Similar results were
obtained with total spleen cells, splenic macrophages, bone
marrow-derived macrophages, and macrophages from the 129 mouse strain
(data not shown).
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+ LPS
treatment induced 20-fold lower p70 compared with p40, and this was
practically abolished by ES-62 treatment (Fig. 3
B).
Interestingly, IL-10 production by peritoneal macrophages was not
detected (sensitivity level of assay <20 pg/ml) in response to ES-62
or any of the stimuli tested over a 72-h time course (results not
shown). Consistent with this, exposure to ES-62, either alone or in
combination with IFN- + LPS, did not induce an increase in IL-10
mRNA levels over a 24-h time period (results not shown).
Intriguingly, the amount of each cytokine produced following ES-62
pretreatment was almost identical in the four experimental groups
(nonstimulated, IFN- alone, LPS alone, and IFN- + LPS; Fig. 3).
This suggests that ES-62 treatment induces low levels of production of
these cytokines before establishing a state of hyporesponsiveness of
macrophages to full activation by IFN- + LPS. Time-course
experiments confirmed that these cytokines are produced during the
ES-62 preincubation (Fig. 4A, and data not shown) but not further stimulated by IFN-
+ LPS (Fig. 4A). These findings were corroborated by
additional studies that showed that ES-62 only induced transient
expression of IL-12 (p40 and p35) and TNF- at the mRNA level as
quantified by real-time PCR (TaqMan) (Fig. 4, B–D).
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+ LPS-induced IL-12 p40 and p35 transcript
levels
To investigate at which level the parasite product is acting to
suppress cytokine production, we tested the effect of pre-exposure to
ES-62 on the subsequent induction of IL-12 and TNF- mRNA by
real-time PCR. Compared with control cells, the level of IFN- +
LPS-induced IL-12 p40 transcripts was reduced by pretreatment with
ES-62 (Fig. 5A). Interestingly, IFN- + LPS stimulated p35 mRNA
expression, but this was also markedly inhibited by pre-exposure to
ES-62 (Fig. 5B). Thus, ES-62 achieves inhibition of
bioactive IL-12 p70 production by targeting both subunits of this
heterodimer. In contrast, TNF- mRNA levels were not significantly
reduced by ES-62 pretreatment (Fig. 5C). We have found that
induction of TNF- production, at both the mRNA and protein levels,
by peritoneal macrophages peaks within 8–10 h post stimulation with
LPS plus IFN- (results not shown). Therefore, these results suggest
that although ES-62 is likely to regulate production of IL-12 p40
predominantly at the mRNA level, it would appear that ES-62 may
modulate TNF- production at a
translational/posttranslational/secretion level.
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To verify whether ES-62 specifically targets production of these
cytokines rather than inhibiting macrophage responses in general, NO
released into the culture supernatant was measured by the Griess
reaction. In contrast to cytokine production, IFN- alone at 100 U/ml
induced NO, whereas LPS (100 ng/ml) had minimal effect. ES-62 had no
effect on NO release from these cells (Fig. 6A). In addition, cell viability was unaffected as determined
by MTT assay (Fig. 6B).
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Exposure of mice to parasite products can be mimicked in vivo
using osmotic pumps, which release their contents at a constant rate.
This offers a more physiological alternative to serial injection. In
agreement with the in vitro data, exposure to ES-62 in vivo (at a rate
of 0.05 µg/h) resulted in the suppression of IFN- + LPS-induced
IL-12 (p40 and p70) and TNF- production (Fig. 7, A and B), while having no effect on NO synthesis
(Fig. 7C). In addition, exposure to ES-62 in vivo did not
result in increased levels of IL-10 production in macrophages
stimulated ex vivo (results not shown). In contrast with the in vitro
exposure, low levels of cytokine production by ES-62-treated but
subsequently nonstimulated macrophages were not readily observed. This
may be due to early and transient cytokine release (Fig. 4), which has
presumably already occurred in vivo. This state of macrophage
hyporesponsiveness appears to be the result of a persistent rewiring of
the cells by in vivo exposure to ES-62 as peritoneal macrophages, which
have been washed and cultured with medium in the absence of ES-62 for
up to 48 h, are still refractory to stimulation with LPS plus
IFN- (results not shown).
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also shows
that the suppression of IL-12 and TNF- production by ES-62 was
dose-dependent (Fig. 7, D and E). Interestingly,
IL-12 production (Fig. 7D) was more substantially inhibited
than TNF- (Fig. 7E) at lower ES-62 concentrations,
whereas NO production was not significantly affected by any of the
treatments (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced
synthesis of IL-12 and, in addition, the proinflammatory cytokines IL-6
and TNF- from murine macrophages. However, the inhibitory effects of
ES-62 are selective in that NO production is not affected. The
relevance of our observations is underscored by the finding that
macrophages recovered from mice exposed to physiological levels of
ES-62 by osmotic pump release in vivo were similarly refractory to
release of IL-12, TNF-, IL-6, but not NO, ex vivo. Thus these
results are entirely consistent with the anti-inflammatory
phenotype associated with patent filarial infection.
ES-62 treatment did not affect macrophage viability, indicating that it
does not suppress cytokine production by simply killing cells.
Furthermore, the ability of macrophages to produce NO was not altered
by treatment with ES-62. The reason for the inhibition of IFN- +
LPS-induced proinflammatory cytokine production in our system remains
to be established. Intriguingly, treatment of macrophages with ES-62
alone induced low levels of IL-12, IL-6, and TNF- production.
LPS-like molecules derived from Wolbachia endobacteria of
O. volvulus can also result in early production of
proinflammatory cytokines such as TNF-, but this is then followed by
anti-inflammatory cytokines such as IL-10 (21). We
found no evidence of IL-10 production in either our in vitro or in vivo
pump-release systems (results not shown). Moreover, IL-10
production following coculture with DO.11.10
CD4+ T cells specific for
OVA323–339 was not enhanced by pretreatment of
the macrophages used as APCs with ES-62 (results not shown). The fact
that ES-62 did not induce macrophage hyporesponsiveness in an
IL-10-dependent manner was rather surprising to us given that previous
reports had suggested that PC-containing moleculesappeared to induce
IL-10 production by B1 cells (22). Moreover, we had
previously shown that ES-62 could induce IL-10 production by dendritic
cells and dendritic-T cell cocultures in vitro (23) and
that IL-10 regulates ES-62-driven IgG class switching in vivo
(17). Although we can find no evidence for IL-10
production in macrophages, the induction of low levels of
proinflammatory cytokines by ES-62 is consistent with an earlier report
that other PC-containing pathogen molecules, zwitterionic
glycosphingolipids of Ascaris suum, induced human PBMCs to
release TNF-, IL-1, and IL-6 (24).
Our data show a two-stage regulation of cytokine production by ES-62:
an initial stimulatory phase during the ES-62 preincubation, followed
by induction of hyporesponsiveness of macrophages to subsequent full
activation by IFN- + LPS. This hyporesponsiveness appears to be the
result of a persistent rewiring of the cells by ES-62 as the
macrophages remain refractory to stimulation with LPS plus IFN- when
cultured for at least 48 h in the absence of ES-62 (Fig. 2 and
results not shown). Interestingly, these findings may mirror our recent
studies that have shown that ES-62 can drive the in vitro maturation of
dendritic cells with the capacity to induce Th2 responses. Such DC
maturation results in increased IL-4 and decreased IFN- production
by DO.11.10 CD4+ T cells in response to challenge
with specific Ag (OVA) relative to that seen with dendritic cells
matured with LPS, a classical stimulator of Th1 responses
(23). Intriguingly, although we have not as yet identified
a molecular mechanism responsible for the development of these
differential DC phenotypes, we have found that although coculture of
ES-62-matured DCs and DO.11.10 CD4+ T cells in
the presence of OVA results in the significant production of bioactive
IL-12 p70, the levels generated are much lower (20–25%) than those
observed in LPS-driven development of Th1 responses (23).
Together, these results may suggest that ES-62 helps drive polarization
of the immune response to a Th2 phenotype by limiting the ability of
APCs to promote the generation of IL-12, a key cytokine in the
development of Th1 responses.
Suppression of cytokine synthesis by ES-62 appears to be due to
modulation of both transcriptional (IL-12) and
translational/posttranslational modification (TNF-). The modulation
of p35 transcript levels by ES-62 is particularly interesting because
p35 has not been as widely studied as its counterpart, p40. Although
constitutively expressed, p35 production can be regulated at multiple
levels, including transcription, translation, and by posttranslational
modification (25, 26). One important mechanism of p35
regulation is the initiation of transcription from different start
sites (25). ES-62 may bias the production of transcripts
that do not encode functional protein, thereby reducing p35 subunit and
bioactive p70 production. In contrast to IL-12, transcription/message
stability of TNF- does not appear to be regulated by ES-62.
Therefore, ES-62 must exert its inhibitory action on the IFN- +
LPS-mediated induction of this cytokine at a later stage of synthesis,
e.g., translation, posttranslational modification, or release from the
cell. For example, the proteolytic cleavage of the membrane-bound form
to release soluble TNF- is an important regulatory step
(27) that may be targeted by ES-62.
Finally, the novel approach of using osmotic pumps to deliver parasite molecules such as ES-62 to mice offers an alternative to repeated dosing by serial injection. Importantly, it mimics natural, constant release of the parasite product during infection, as well as minimizing animal handling and stress, which can influence the immune response. Because measurement of ES-62 levels in the bloodstream of the mice by sandwich ELISA showed them to be at the lower end of levels of PC-containing molecules circulating in infected humans (results not shown), these experiments confirm the physiological relevance of our in vitro data. Thus, exposure to ES-62 during natural filarial nematode infection is likely to inhibit macrophage responsiveness and subsequent Th1 polarization.
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Foo Y. Liew, Department of Immunology and Bacteriology, University of Glasgow, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, U.K. E-mail address: f.y.liew{at}clinmed.gla.ac.uk
3 Abbreviations used in this paper: ES, excretory-secretory; PC, phosphorylcholine; FAM, 6-carboxy-fluorescein; TAMRA, 6-carboxy-tetramethyl rhodamine; m, murine; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
Received for publication February 21, 2001. Accepted for publication May 14, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or preincubated with 2 µg/ml ES-62 for 18 h
(
) before stimulation with IFN-
) for 18 h and then stimulated with
IFN-
